Abstract

The protozoan parasites Leishmania, Trypanosoma cruzi and Trypanosoma brucei show multiple features consistent with a form of programmed cell death (PCD). Despite
some similarities with apoptosis of mammalian cells, PCD in trypanosomatid protozoans
appears to be significantly different. In these unicellular organisms, PCD could represent
an altruistic mechanism for the selection of cells, from the parasite population,
that are fit to be transmitted to the next host. Alternatively, PCD could help in
controlling the population of parasites in the host, thereby increasing host survival
and favoring parasite transmission, as proposed by Seed and Wenk. Therefore, PCD in
trypanosomatid parasites may represent a pathway involved both in survival and propagation
of the species.

Programmed cell death in trypanosomatids

The unicellular protozoan parasite Leishmania, Trypanosoma cruzi and Trypanosoma brucei are the causative agents responsible for human leishmaniasis, Chagas disease, and
African sleeping sickness, respectively. These trypanosomatid parasites have complex
life cycles that involve multiple hosts. Inside the insect vector or mammalian host,
these parasites undergo differentiation and multiplication phases. By some unknown
signals/mechanisms, some individuals in the total population differentiate into non-dividing
but infectious forms, predisposed to live inside the next host. However, the fate
of the remaining non-infectious forms is not known. Are these cells eliminated by
a process akin to programmed cell death?

Recently, several reports have provided evidence for a form of programmed cell death
(PCD) in the trypanosomatid parasites Leishmania, Trypanosoma cruzi and Trypanosoma brucei [reviewed in ref. [1]]. We and others have shown that several features of PCD described in higher eukaryotes
were also found in these trypanosomatid parasites [reviewed in ref. [1]]. These features include depolarization of mitochondrial membrane potential, release
of cytochrome C, activation of proteases, phosphatidyl serine exposure, loss of plasma
membrane integrity and DNA fragmentation. Such features have been observed in parasites
in culture in vitro either upon treatment with various stimuli i.e. H2O2, staurosporine, amphotericin B, or in late stationary phase cultures [reviewed in
ref. [1]]. Further, some of the regulatory and effector molecules that have similarity to
known PCD factors in either higher eukaryotes or unicellular organisms also have been
identified in trypanosomatids [reviewed in ref. [1]]. In addition, evidence of PCD has also been observed in vivo e.g. in dying T. brucei rhodesiense inside the midgut of tsetse flies [2] or in Leishmania donovani amastigotes inside macrophages isolated from patients that were treated with antileishmanial
drugs [3].

Although some similarities with apoptosis of mammalian cells can be drawn, it is believed
that the type of PCD observed in unicellular trypanosomatids is different from that
described in higher eukaryotes [[1], references there in]. Whether these differences reflect the remains of an ancient
form of PCD in trypanosomatids that evolved into the sophisticated apoptosis process
in mammalian cells or the necessary adaptation of unicellular parasites to survive
inside their various hosts remains to be elucidated. The important question still
remaining is what could be the role of PCD in trypanosomatid parasites. In order to
address this question, it is fair to assume that if unicellular parasites have retained
such a PCD pathway during evolution, it is because this pathway must be beneficial
or essential for survival of the species or population.

In the case of Leishmania, inside the gut of the sandfly vector, the procyclic promastigote forms of the parasite
multiply and differentiate into several intermediate forms and ultimately into infectious
metacyclic promastigotes. Only the infectious metacyclics are transmitted into the
mammalian host and have the ability to successfully differentiate into amastigotes
and establish an infection. Since not all the promastigotes differentiate into metacyclic
forms inside the gut of the insect, we believe that the remaining procyclic forms
may undergo PCD, as seen in stationary phase culture in vitro [1]. Such an elimination process of procyclic forms would be beneficial for the rest
of the population (i.e. metacyclics) since these parasites would not utilize the limited
supply of essential nutrients such as purines or heme (that they are not able to synthesize
de novo) present in the insect gut. Therefore, PCD in the Leishmania promastigote stage may represent an altruistic mechanism for the selection of parasites
that are fit to transmit the disease to the mammalian host, which is a critical step
for the propagation of parasites.

Inside the mammalian host, there is a slow multiplication phase of Leishmania amastigotes (over weeks in the hamster model, or years in the case of human visceral
leishmaniasis) leading to an accumulation of infected macrophages in the spleen and
liver. During this chronic phase of the disease, one can assume a continuous release
of amastigotes in the infected organs due to the bursting of infected macrophages.
The free extracellular amastigotes are then phagocytosed by new macrophages inside
which they multiply. This reinfection cycle occurs without triggering an overwhelming
immune response by the host, which would be detrimental for the parasite survival.
PCD might play a role in this silencing of the host immune response since recent findings
showed that uptake of apoptotic T lymphocytes by macrophages infected with T. cruzi increases parasite growth inside these macrophages [4]. Such uptake of apoptotic T cells renders the macrophages refractory to inflammatory
cytokines, a process probably mediated by transforming growth factor-beta production,
and allowing parasite survival and growth. Since L. donovani amastigotes (axenically grown) can enter a PCD pathway [1], it is possible that "apoptotic-like" amastigotes in vivo could also play a role
in silencing the host immune response when they enter the macrophages along with non-apoptotic
amastigotes. Such a process could maintain the parasite growth in the infected tissues
during the chronic phase of the disease. Further, this increased survival of parasites
through the programmed sacrifice of some would improve the probability of completing
their life cycle through the bite of a sandfly and ultimately resulting in the propagation
of the species.

Such an altruistic behavior of a trypanosomatid parasite is also proposed by Seed
and Wenk in this current issue. These authors argue that the transition from long
slender (LS) to short stumpy (SS) forms of African trypanosome parasites in the mammalian
host has evolved in part to help control the parasitemia and to increase host survival
time. These authors show that after transformation from LS to SS, only a fraction
of SS parasites undergo apoptotic-like events, which lead to their cell death and
their stimulation of the host immune system. Unlike Leishmania, stimulation of the host immune system by SS trypanosomes undergoing PCD leads to
elimination of most of the parasite population from the host and selection of minor
LS variant and new infective SS forms, which favor parasite transmission for a longer
time by keeping the host alive.

In conclusion, in order to successfully demonstrate the real purpose of the PCD, whether
altruistic or otherwise, in Leishmania and other trypanosomatid parasites, it is essential to first establish PCD in vivo,
in an infected host. In addition, the molecular characterization of effector molecules
that are critical for the parasite PCD pathway is essential because such process could
be exploited for novel therapeutic intervention.